Project Summary/Abstract Tissue factor (TF) activation on the cell surface must be exquisitely regulated to ensure hemostasis following injury but prevent pathologic thrombosis. Inappropriate TF procoagulant activity underlies substantial human suffering, including that due to myocardial infarction, venous thromboembolism, stroke, and sickle cell disease. Despite 40 years of investigation, the mechanisms by which TF is activated on membrane surfaces remain to be proven. We revisit this fundamental problem in an unbiased fashion by performing a genome-wide screen to identify new modifiers of TF cell surface expression and procoagulant activity. We hypothesize that what has been termed ?encryption/decryption? of TF is a highly regulated event involving multiple activation pathways rather than a single dominant mechanism. As TF initiates blood coagulation in vivo, a complete understanding of TF activation has broad implications for hemostasis and thrombosis. In Aim 1, we will further evaluate the 21 positive and 114 negative regulators validated in the screen to identify the subset that post- transcriptionally regulate TF procoagulant activity, including those acting directly on the TF cytoplasmic tail, and employ bioinformatics strategies to define physiologically important pathways and networks regulating TF. Among these, our preliminary data identify S-acyltransferase activity as a highly enriched gene set regulating TF coagulant activity, and a specific palmitoyltransferase ZDHHC2 that suppresses procoagulant activity to nearly the extent of tissue factor pathway inhibitor, the strongest known negative regulator of TF. In Aim 2, we will evaluate the contribution of ZDHHC2 to TF encryption and TF cytoplasmic tail phosphorylation, and test the determinants of TF S-acylation using palmitate-alkyne labeling and click chemistry. In Aim 3, we will use a novel CRISPR/Cas9 mouse that lacks the evolutionarily conserved cysteine in the TF cytoplasmic tail and, in parallel, Zdhhc2 null mice to evaluate the role of TF palmitoylation during thrombus formation in vivo. This proposal describes a five-year research and training program for Dr. Sol Schulman's mentored career development. The applicant previously completed MD-PhD training in the medical scientist training program at Harvard Medical School and is committed to a career as a physician-scientist in academic hematology. The division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center has a distinguished track record of scientific innovation and mentorship, overall affording an excellent environment for the applicant to advance his research and career goals. Drs. Robert Flaumenhaft and Bruce Furie will serve as the primary mentor and co-mentor, respectively, and a senior advisory committee will bring diverse intellectual expertise to Dr. Schulman's efforts. After this comprehensive program, the applicant will be well positioned to succeed as an independent investigator in the field of hemostasis and thrombosis.